A new combination of RT-PCR and reverse dot blot hybridization for rapid detection and identification of potyviruses

Hsu, Yueh-Chwen; Yeh, Tzu-Jung; Chang, Yuan-Chi

doi:10.1016/j.jviromet.2005.04.002

Cited by 34 publications

(20 citation statements)

References 22 publications

(27 reference statements)

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“…According to our knowledge this approach has not been applied by other researchers to detect that virus. Dot blot hybridization using nucleic acid probes has been used by several research groups to detect and diagnose other plant viruses in the world, and its advantages were established [18,30,32,38,40] stated that plant virus identification and characterization can be accomplished by several methods involving their morphological, physical, biological, cytological, serological and molecular properties. The use of molecular techniques is increasing worldwide, and some have been developed for identification and characterization of plant viruses.…”

Section: Resultsmentioning

confidence: 99%

Biological, Serological and Molecular Characterization of Papper MildMottle Virus Isolated from Weast Region of Kingdom of Saudi Arabia

Alwabli¹,

Khattab²,

Farag³

2017

Res J Infect Dis

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In this paper, The virus was identified based on host range, symptomatology, and stability in crude extracts, modes of transmission, serological reaction, inclusion bodies, electron microscopy and molecular biology. Study ofthe host range which inoculated different plant species belonging to twelve families revealed that the reactions different hosts. Data concerning stability of virus in crude sap were TIB95C 0 ,DEP10 -7 -10 -8 and LIV 28 days. Modes of transmission showed that PMMoV transmitted mechanically and by infected seeds but cannot be transmitted byany of the tested aphid species.A one-step reverse-transcription polymerase chain reaction (RT-PCR) assay was used for the detection and identification of the isolated virus from nucleic acid extracts of infected pepper plants .Using specific oligonucleotide primer PMMoV-F/ PMMoV-R, which amplified the coat protein (CP) gene for detection of Pepper mild mottle virus (PMMoV). A major product of approximately 387bp PMMoV-CP gene was produced. DNA-DNA hybridization assays of viral genome have been used for detection of the present virus isolate using specific cDNA probe prepared for PMMoV. A one-step reverse-transcription polymerase chain reaction (RT-PCR) assay was used for the detection and identification of the isolated Pepper mild mottle virus (PMMoV) from nucleic acid extracts of infected pepper plants.Using specific oligonucleotide primer PMMoV-F/PMMoV-R, which amplified the coat protein (CP) gene for detection of Peppermild mottle virus (PMMoV). A major product of approximately 387bp PMMoV-CP gene was produced. A high reliable sensitive immunocapture reverse transcriptionpolymerase chain reaction (IC-RT-PCR) technique was applied for detection of PMMoV in which the in infected pepper plant. PCR fragment of correct size 387bpwas amplified with primer specific coat protein gene. DNA-DNA hybridization assays of viral genome have been used for detection of the present virus isolate using specific cDNA probe prepared for PMMoV. This study of PMMoVis carried out for the first time in Taif Saudia Arabia.

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Section: Resultsmentioning

confidence: 99%

Biological, Serological and Molecular Characterization of Papper MildMottle Virus Isolated from Weast Region of Kingdom of Saudi Arabia

Alwabli¹,

Khattab²,

Farag³

2017

Res J Infect Dis

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“…A part of the motif, X-T-X-X-X-N-T-(32 amino acids)-G-D-D, is also present in the SLRMV-NSV9 polyprotein at amino acid positions 1-42. In particular, the first six amino acids (S-T-V-V-D-N) of the SLRMV-NSV9 polyprotein are completely identical to the G-N-N-S-G-Q-P-S-T-V-V-D-N sequence, that is highly conserved among potyviruses (Hsu et al 2005).…”

Section: Complementary Dna Amplification and Sequence Analysesmentioning

confidence: 98%

Soybean leaf rugose mosaic virus, a new soilborne virus in the family Potyviridae, isolated from soybean in Japan

et al. 2010

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In 2005, soybean plants with severe mosaic and leaf rugose symptoms were found in Japan. The plants contained flexuous and filamentous virus-like particles with a modal length of 500 nm. Double-stranded RNA analysis revealed a single major band of *7000 bp. Reverse transcription-PCR for Potyviridae diagnosis generated a 1.5-kbp cDNA fragment, which contained a partial NIb gene, a coat protein (CP) gene, a 3 0 -noncoding region, and a poly(A) tract. The CP gene consisted of 248 amino acids and had less than 40% amino acid sequence identity with CPs of other member in Potyviridae. The C-terminal region of the NIb gene had *60% amino acid sequence identity with Barley mild mosaic virus and other Bymovirus isolates. The NIb amino acid sequence contained the GDD motif, while the CP had no motifs associated with aphid transmission. Phylogenetic analyses of the amino acid sequence of the CP core region showed that this virus was independent from other potyvirus genera. In a host range test, the virus infected 10 plant species in four families. The virus was not transmitted via seeds or aphids, but soybeans became diseased when grown in virus-infested soil. The characteristics of this virus suggest that it is not related to any of the previously described soybean viruses; thus, we tentatively propose the name soybean leaf rugose mosaic virus (SLRMV).

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“…The firststrand cDNA was synthesised using dT-Bam (5′-AGCTGGATCC(T) 18 -3′) or mtR1 primers. PCR reactions were carried out using dT-Bam and PNIbF1 (5′-GGBAAYAATAGTGGNCAACC-3′) primers for potyviruses (Hsu et al 2005), or mtR1/mtF2 primers for plant mitochondrial nad5 gene gene. RT-PCR products were analysed in 1% agarose gels and the desired cDNA fragments were purified by GFX TM PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).…”

Section: Primer Designmentioning

confidence: 99%

Detection of four calla potyviruses by multiplex RT-PCR using nad5 mRNA as an internal control

Huang

Lee

et al. 2009

Eur J Plant Pathol

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Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Konjac mosaic virus (KoMV) and Zantedeschia mild mosaic virus (ZaMMV) are important potyviruses previously identified in calla lily plants in Taiwan. In order to save time and cost of virus detection, a multiplex RT-PCR assay was developed for these calla potyviruses. Specific primers for each virus were designed based on the sequences of 3′ terminal region of respective viruses. To prevent false negative results, a primer pair specific to plant mitochondrial nad5 mRNA was used to produce a 185-bp fragment as an internal control of RT-PCR. The specificities of primers were confirmed by means of simplex and multiplex PCR assays. Optimal primer concentration ratio was identified by multiplex PCR assay. Total RNAs purified from virus-infected plants were used directly or mixed in different combinations, and then tested by multiplex RT-PCR. The result indicated that the expected RT-PCR products could be specifically amplified and identified on the basis of their molecular sizes. The detection sensitivity of multiplex RT-PCR was 25-625 times higher than that of indirect-ELISA (I-ELISA) depending on the virus. When applied to field surveys, multiplex RT-PCR could detect more single as well as mixed infection samples than I-ELISA. Accordingly, our multiplex RT-PCR assay provides a simple, rapid and reliable method for multiple potyvirus detection in calla lily.

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A new combination of RT-PCR and reverse dot blot hybridization for rapid detection and identification of potyviruses

Cited by 34 publications

References 22 publications

Biological, Serological and Molecular Characterization of Papper MildMottle Virus Isolated from Weast Region of Kingdom of Saudi Arabia

Biological, Serological and Molecular Characterization of Papper MildMottle Virus Isolated from Weast Region of Kingdom of Saudi Arabia

Soybean leaf rugose mosaic virus, a new soilborne virus in the family Potyviridae, isolated from soybean in Japan

Detection of four calla potyviruses by multiplex RT-PCR using nad5 mRNA as an internal control

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